P2-091 Neuronal loss in selected brain areas of a new transgenic AD rat model estimated with unbiased stereological methods

P2-091 Neuronal loss in selected brain areas of a new transgenic AD rat model estimated with unbiased stereological methods

Poster Session P2: Animal and Cellular Models - Animal Models, Transgenic ~ SEVERE LEARNING AND MEMORY DEFICITS AND PRONOUNCED Afl-HISTOPATHOLOGY IN...

322KB Sizes 0 Downloads 5 Views

Poster Session P2: Animal and Cellular Models - Animal Models, Transgenic

~

SEVERE LEARNING AND MEMORY DEFICITS AND PRONOUNCED Afl-HISTOPATHOLOGY IN A TRANSGENIC MOUSE MODEL CONTAINING A TRIPLE MUTATED HUMAN APP (AMYLO1D PRECURSOR PROTEIN)

Gueuther Ranter*, Manuela Wedenig, Daniel Havas, Maria Posch, Ulrike Bauer, Edith Schreiner, Birgit Hutter-Paier, Manfred Windisch. JSW-Research, Graz, Austria. Contact e-mail: [email protected] Alzheimer's disease (AD) is characterized by amyloid plaques and neurofibrillary tangles as well as progressive neuronal degeneration. Missense mutations in the APP gene, which can influence the A~ cleavage and lead to a higher aggregation potential are related to Familial Alzheimer's disease (AD). For the present study we established a new transgenic mouse model on a pure C57BL/6 background bearing human (h) APP751 (amyloid precursor protein 751) containing the London (V7t7I), the Swedish (K670M/N671L) and the Arctic (E693G) mutations under the regulatory control of the tissue-specific murine (m) Thy-1 promoter (mThy-l-hAPP751). Our data show high expression of the transgene on mRNA as well as on protein levels. Western blot analysis indicates an APP processing which is different compared to a mouse model carrying the London and the Swedish mutation alone. Cognitive abilities of two lines (TLSA14 and TLSA26) were tested in the Morris water maze with an age of 7 and 15 months. Results show severe deficits in learning and memory already with an age of 7 months and impairments were even pronounced with 15 months. First immunohistochemical investigations with the hA[3-specific antibody 6El0 show clear [3-amyloid pathology in the frontal cortex and in the hippocampus. The study will be continued with extensive analyses including neuronal cell death, evaluation of APP processing and generation of Af3-fragments and cytoskeletal changes (neurofilament, tan, MAP2). The first results indicate that we generated a valid model for neurodegeneration and therefore a new seminal AD mouse model.

P•-•

EARLY BEHAVIORAL ABNORMALITIES IN A MOUSE MODEL OF FTDP-17

Yoshitaka Tatebayashi*, Takashi Tominaga, Tatsuya Mizoroki, Emmanuel Planel, Naruhiko Sahara, De-Hua Chui, Akihiko Takashima. Brain Science Institute, RIKEN, Wako-shi, Saitama, Japan. Contact e-mail: marsiny @brain, riken, go.jp

Background: Tan gene mutations cause frontotemporal dementia and parkinsonism linked to chromosome-17 (FTDP-17) in humans. Although tauopathy is one of the major consequences of such mutations, the relationship between behavioral and neuropathological changes in vivo have been elusive. Objective(s): Early behavioral changes were investigated in a mouse model of FTDP-17. Methods: We generated transgeuic (Tg) mice expressing modest levels of the longest human tan isoform with or without one of FTDP-17 tau gene mutations (R406W) under the control of c~-CaMKII promoter. This mutation generally causes Alzheimer-type dementia and tauopathy in humans. We have studied these mice (3 ~ 21 month-old) behaviorally (forced swim test, fear conditioning test, etc.), electrophysiologically (LTP), and neuropathologically. Results: Exogenous human tan was expressed mostly in axons of postnatal forebrain neurons in Tg brains. R406W but not wild-type (WT) tan Tg mice exhibited significant behavioral changes associated with emotion, fear and memory even when they were young (as early as 7 month of age). Male mice exhibited earlier onset of these abnormalities. Such abnormalities appear to be functional because no apparent neuropathological change was found in these young mice. Conclusions: We found that axonal expression of modest levels of mutant human tau in mice causes functional behavioral abnormalities in mice. This finding may provide insight into the early diagnosis and prevention of the disease.

~

$249

ABETA42 IS REQUIRED FOR AMYLOID DEPOSITION IN TRANSGENIC M I C E

Fiona Pickford *1 , Luisa Onstead-Cardinale t, Jason Eriksen 1, Paul Murphy 1, Das Pritam I Jungsu Kim 1, Georgia Dolios 2, Rong Wang 2, Dennis W. Dickson ~, Todd Gotde 1, Eileen McGowan I . 1Mayo Clinic, Jacksonville, FL, USA,"2Mount Sinai School of Medicine, New York, NY, USA. Contact e-mail: [email protected]

Background: Current transgenic mouse models that deposit A{3 in the brain as plaques, also overexpress human mutant APE Thus, any study of the role of A[3 deposition in AD using current transgenic mice is potentially confounded by overexpression of APR At present there are no animal models that generate A[3 in the secretory pathway, in the absence of APP overexpression, at levels sufficient to cause amyloid pathology. Objective(s): To determine whether A[340 or AI342 are sufficieut and/or necessary to develop amyloid type pathology in transgenic mice. Methods: Transgenic mice that produce either A[340 or A[342 peptides were generated by expressing specific BRI-A~ fusion constructs under control of the mouse PrP promoter. Brain A[3 levels were analyzed by sandwich ELISA. The A[3 peptides generated in vivo were determined by mass spectroscopy and the development of pathology was assessed in a series of mice from 3-20 months of age. Results: Mass-spectroscopy analysis of brain tissue from BRI-A{342 and BRI-A[342 confirmed the production of A[M0 and A1342 in these mice, respectively. There was no evidence of A[3 truncation in either transgenic model. BRI-A[342 mice had elevated brain A[3 levels at 3 months of age, with age related increases in both soluble and formic acid extracted A[3. By 5-6 months both fibrillar and diffuse amytoid deposits were present in the cerebellum of the BRI-A[342 mice. At older ages BRI-A[342 mice developed extensive pathology in the hippocampus and cortex and congophilic amyloid angiopathy. There was no evidence of overt cell loss. Although, young BRI-A[340 mice typically had higher levels of brain A[340 than young Tg2576 mice there was no immunohistochemical or biochemical evidence of A[3 accumulation in these mice at ages up to 20 months. Conclusions: Increased A{340 expression may not be sufficient to cause amyloid deposition, however, overproduction of human Af342 is, by itself, sufficient to seed amyloid deposition. Expression and deposition of AI342 in the absence of APP does not result in widespread neurodegeneration. These mice are currently being used to examine the respective roles of A[340 and A[342 in seeding A[3 deposition, plaque growth and other pathology.



NEURONAL LOSS IN SELECTED BRAIN AREAS OF A N E W TRANSGENIC AD RAT MODEL ESTIMATED

WITH UNBIASED STEREOLOGICAL METHODS Peter Koson* 1, Norbert Zilka I , Peter Filipcik 2, Michal Novak 1,2. t Institute of Neuroimmunology, Slovak Academy of Sciences, Bratislava, Slovakia, Bratislava, Slovakia; 2Axon-Neuroscience GmbH, Vienna, Austria. Contact e-mail: [email protected] savba.sk

Background: Alzheimer's disease (AD) is neurodegenerative disorder accompanied with loss of principal neurons. Previous studies investigating the extent of neuronal loss have demonstrated a statistically significant decrease of pyramidal neurons in the human hippoeampus, especially in the CA 1 area (West, 1994). Furthermore histopathological correlation was demonstrated between neuronal loss, Braak-stageing of AD and the amount of hippocampal neurofibrillary tangles (Rrssler, 2001). Objective: To determine neuronal loss in selected brain areas of Axon's new Alzheimer's rat model in correlation with the amount of neurofibrillary tangles using unbiased stereological methods - optical fractionator. Methods: The study on transgenic animals and age-matched controls was realized with the aid of computer-based stereological system (StereoIm,estigator 5.05.4, MicroBrightField, Vermont, USA). Number-weighted variation of the optical fractionator was applied on Nissl-stained sections for estimation of neuronal cell numbers and on Gallyas-stained sections for estimation of NFTs amount. The study was carried out as "blind" study. Results: The amount of principal neuron loss in the hippocampus and in selected reticular nuclei of midbrain, pons and medulla oblongata was determined. We found that our new AD transgenic rat model exhibits massive neurofibriltary degeneration in cortex,

$250

Poster Session P2: Animal and Cellular Models - Animal Models, Transgenic

hypothalamus, midbrain, pons, cerebellar nuclei, medulla oblongata and in the spinal cord. Progressive increase of NFTs was observed with increasing age of transgenic animals. Stereological estimations of the most vulnerable brain areas showed decreased neuron counts. Conclusions: Analysis of Axon's new transgenic AD rat model showed that reduction of neuron numbers in the most affected brain areas correlates with progression of NFT development.

~

ABERRANT ACTIVATION OF CDK5 TRIGGERS EVENTS LEADING TO ALZHEIMER'S PATHOLOGY

Jonathan C. Cruz*, Li-Huei Tsai. Harvard Medical School, Boston, MA, USA. Contact e-mail: [email protected] Background: Cyclin dependent kinase 5 (Cdk5) and its regulatory subunit p35 are integral players in the proper development of the mammalian central nervous system. Various neurotoxic conditions, including [3-amyloid (A[3) treatment, lead to the proteolytic cleavage of p35 by calpain into a truncated p25 species. Upon binding to this pathogenic p25 activator, Cdk5 is deregulated. Aberrant Cdk5 activity has been implicated in several neurodegenerative diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, and Niemann-Pick type C. In all of these cases, the increase in Cdk5 activity is accompanied by the accumulation of the toxic p25 activator. Overexpression of p25 in cultured cortical neurons results in disruption of cytoskeleton, hyperphosphorylation of tan, and apoptotic cell death, indicating that aberrant Cdk5 activation is neurotoxic. Recent evidence from cultured neurons also suggest a role for Cdk5 in modulating amyloid precursor protein (APP) metabolism. Objective(s): The aim of this study was to test the hypothesis that in vivo pathogenic activation of Cdk5 by p25 is a causative factor in progression of the pathological hallmarks associated with Alzheimer's Disease, including neuronal loss, neurofibrillary tangle pathology, and abnormal APP processing. Methods: The tetracycline-controlled transactivator system was utilized to generate hitransgenic mice that inducibty overexpress human p25 under the control of the CamKIl promoter, which drives high transgene expression in the forehrain. Conclusions: The resulting increased Cdk5 activity in the p25 transgenic mice led to progressive neuronal loss in the cerebral cortex and hippocampus, which was accompanied by severe forebrain atrophy, reactive astrogliosis, and caspase-3 activation. Endogenous tan was also hyperphosphorylated at many epitopes, aggregated tan fibrils accumulated, and neurofibrillary pathology developed progressively in these mice. Furthermore, the processing of endogenous APP was altered in the transgenic mice, resulting in increased A[~ generation, which was detected prior to tau pathology in the mice. Our findings provide a novel link between Alzheimer's pathology, including neuronal loss, tau, and APP pathology, and p25 deregulated Cdk5 activity. They also highlight the importance of protein phosphorylation in the progression of neurodegenerative diseases and provide an impetus to develop therapeutic strategies to specifically inhibit p25 accumulation and aberrant Cdk5 activation.

~

M E T A B O L I C M A P P I N G O F GLUCOSE UPTAKE IN

PSAPP MOUSE MODEL OF AD: SENSORIMOTOR HYPERAROUSAL CORRELATED WITH AUDITORY AMYLOID PATHOLOGY Lonnie E. Schneider* 1,2, Jon Valla 1'2, Eric M. Reiman 32 . 1Barrow Neurological Institute, Phoenix, AZ, USA; 2Arizona Alzheimer's Disease Consortium, Phoenix, AZ, USA; 3Dept of Psychiatry, University of Arizona, Tucson, AZ, USA. Contact e-mail: [email protected] Background: While transgenic mice have great promise in the study of Alzheimer's disease (AD), uncertainties remain about the extent to which they provide a model of the disorder or the best way to characterize disease progression. Using fluorodeoxyglucose (FDG) autoradiography, we found that very old homozygous PDAPP transgenic mice have preferentially and progressively reduced activity in the posterior cingulate/retrosplenial cortex, as in FDG PET studies of persons affected by or at genetic risk of AD. However, the PDAPP mice have a significant white matter abnormality

(i.e., a truncated corpus callosum), which may preclude their use in in vivo metabolic imaging studies due to potentially confounding partial-volume effects. If other transgenic models of AD demonstrate significant declines in PCC activity in the absence of this white matter abnormality, it may be possible to track the progression of the PCC abnormality and screen candidate treatments more efficiently. Objective(s): To replicate our earlier fnding of abnormally low PCC metabolism in double-transgenic PS lxTG2576APP (PSAPP) mice, which have significantly less pronounced white matter abnormalities. Methods: FDG autoradiography followed by anatomic densitometric imaging, was used to determine glucose uptake in various regions in PSAPP (n = 12) and littermate controls (n = 13) at approximately 16 months of age. A 2 x 2 between(group)-within(hemisphere) ANOVA was used to evaluate the effects of transgene by hemisphere in the mice. Results: PSAPP transgenic mice showed significant reductions in FDG uptake in retrosplenial cortex and anteroventral thalamus. They also showed significant alterations in several additional regions underlying arousal (reticular thalamus, periaqueductal gray) and sensorimotor function (ventromedial and ventrolateral thalamus, subthalamus, cerebellum). The PSAPP mice also showed significant increases in the primary auditory pathway (inferior colliculus, medial geniculate, primary cortex) coincident with pronounced and circumscribed amyloid pathology in these nuclei. Conclusions: We continue to explore the extent to which metabolic mapping measurements in PSAPP transgenic mice and other putative animal models of AD could be used to help clarify disease mechanisms and screen candidate treatments.

•P•--•

CELLS INVOLVED IN F I B R I L L A R AMYLOID DEPOSITION IN TRANSGENIC M I C E W I T H A H U M A N A P P - T R I P L E MUTATION

Janusz Frackowiak*, Richard Rubenstein, Jerzy Wegiel, Giuseppe LaFauci, Humi ImaM, Kuo C. Wang, Wojciech Kaczmarski, Thirasak Snkontasup, Dagmar Kowal, Bozena Mazur-Kolecka. NYS Institute for Basic Research in Developmental Disabilities, Staten Island, NY,, USA. Contact e-mail: [email protected] omr.state.ny, us

Background: Human APP transgenic mice in which brain deposition of amyloidq3 takes place, are widely used as animal models of Alzheimer's amyloidosis-[L Characterization of the role of brain cells in production and fibrillization of amyloid-[3 in animal models may determine the mechanisms of parenchymal and vascular amyloidosis-[~ in human brain. Objectives: The aims of this study were (1) to generate transgenic mice in which all three forms of human brain amyloidosis are reproduced, including fihrillar plaques, amyloid deposits in capillaries and arteries and veins; (2) to characterize the mechanisms of formation of these three types of lesions. Materials and Methods: Three strains of mice transgenic with human APP with three mutations: Swedish, Dutch and London, expressed under the PrP promoter, were generated. The transgene expression was characterized by PCR. Production of APP and A[3 were determined by immunoblotting. The onset, topography and amount of parenchymal and vascular amyloid were determined by immunohistochemistry. The relationships between the cells and amyloid deposits were examined at the ultrastructural level. Results: The strain Tg9291 had a high number of transgene copies, more than 30 per genome. Brain levels of AIM-40 and AIM-42 were 6 fmol/100 t~g protein, in 6-week-old animals. At the age of 12 weeks numerous fibrillar amyloid plaques and vascular amyloidosis were detected in all regions of cortex and in hippocampus in Tg9291 mice. In the strain Tg9279, brain levels of A[~ were lower (A~I-40 was 3 fmol/100 Ixg protein, AIM-42 was 2 fmol/100 Izg protein, in 6-week-old animals), and fibrillar amyloid deposits appeared much later, at 6 months of age. No A[~ deposition was found in the strain Tg9294, with the lowest A~ brain levels, up to 9 months of age. The morphological studies indicate that the ceils of the monocyte-macrophage lineage play a critical role in fihrillar amyloid deposition, both in the wall of capillaries and in parenchymal plaques. Smooth muscle cells are involved in deposition of amyloid in tunica media of arteries. Condusions: Fibrillar amyloid-[3 deposition in APP-Swe-D-Lon transgenic mice recalls the mechanisms of fibrillar plaque formation and amyloid angiopathy in human brain.